The development of prophylactic vaccines against HIV is of paramount importance in the global drive to halt the spread of the virus. Most new vaccine candidates are based on (or contain) versions of the envelope glycoprotein (Env), the target of the desired broadly neutralizing antibodies. However, even after the successful discovery and initial testing of a vaccine candidate, there are hurdles associated with production yield, purification strategy and in vitro stability that may hinder its development as a therapeuti compound. In this proposal, we aim to optimize the vaccine development pipeline in order to speed up the process of getting promising leads to the clinic. In a partnership between a research university (University of Puerto Rico), a biotechnology start-up (CDI laboratories) and the advisory input of local biopharmaceutical partners (Amgen Manufacturing, Eli Lilly), we will apply state-of-the-art analytical tools to optimize the production of trimeric gp145, a promising new vaccine candidate discovered and partnered between the Henry M. Jackson Foundation for Military Science and the NIH Vaccine Research Program. It has been shown by our group and by others that the yields of trimeric gp145 expressed in CHO cells, are consistently low (3-4 mg/L) and that informative analytical methods have not been developed to measure reagent quality or to predict its efficacy. Thus, we propose to develop a suite of analytical tools to quicly assess glycan composition, aggregation, binding activity and protein degradation (Aim #1). We will then implement these analytical tools to the process of clone selection (Aim #2) and to the optimization of protein expression conditions (Aim #3). Finally, we address a long-standing claim that the low yields of trimeric gp145 are due to its toxicity toward the production host, CHO-K1 (Aim #4). The proposed work is a continuation of efforts in our group involving this particular vaccine candidate. Our group has already developed a MALDI-ToF mass spectrometry method for the measurement of glycan composition of trimeric gp145 and we have used the resulting information to evaluate the effect of modifying the media composition on glycan distribution. We have developed methods for the detection of protein aggregates using dynamic light scattering and for the quantification of protein binding using a biosensor assay. For the first time, we have also shown that exogenously added trimeric gp145 to CHO-K1 cells, dramatically compromise their viability in culture, thus suggesting potential toxicity issues that are best addressed during the early stages of vaccine development. All proposed analyses of gp145 will be performed using appropriately qualified methods to facilitate eventual transfer of all methods into manufacturing facilities. In summary, the experimental tools developed in this proposal will help to make better decisions on the production of trimeric gp145, potentially boosting the production yields and quality en route to clinical trials.